Human Axl APC-conjugated Antibody

Detection of Human Axl by Flow Cytometry

CD1c+ DCs in LPS-BAL are likely to be tissue-recruited blood CD1c+ DCs. a Expression of surface antigens by flow cytometry on DC2/3 from HC blood (green), SS-BAL (blue) and LPS-BAL (red) relative to isotype control (gray). Antigens predicted to discriminate between blood and tissue CD1c+ DCs were tested. Representative plots from more than three experiments are shown. b Dendrogram showing hierarchical clustering of monocyte/macrophage and DC gene expression by indicated subsets isolated from HC blood and SS/LPS BAL. In vitro monocyte-derived DCs were also included. Clustering used Pearson correlation metric. Monocyte/macrophage and DC identifying genes were taken from McGovern et al.28. The 25 genes available on the NanoString Immunology v2 panel were used. c Proliferation of allogeneic peripheral blood T cells measured by CSFE dilution during 7-day co-culture with or without MP subsets isolated from LPS BAL. Flow cytometry plots are gated on CD3+ T cells from a representative experiment. Summary graph shows 2–3 replicates per subset. Bars show mean ± SEM. Means were compared by one-way ANOVA with Dunnett’s multiple comparison test of DC2/3 against other subsets. *p < 0.05. d Analysis of differentially expressed genes (DEGs) by DC2/3 from SS/LPS-BAL and HC blood. Comparisons were made by unpaired t-test with p < 0.05 and >3-fold difference in mean expression. Venn diagram shows DEGs in DC2/3 from SS-BAL, LPS-BAL, and HC blood. Circles represent DEGs between SS BAL and blood (cyan; 154 genes, 33%) and between LPS-BAL and blood (yellow; 124 genes, 39%). The overlapping circles represents DEGs shared between comparisons (100 genes, 21%). The top 10 upregulated genes (standard type) and top 5 downregulated genes (italic type) in BAL relative to blood are listed. Heatmap shows DEGs common to DC2/3 in both SS-BAL and LPS-BAL relative to HC blood. Genes with >10-fold difference in mean expression are shown. Genes discussed in the text are colored burgundy. e Heatmap showing DEGs between DC2/3 from SS-BAL and LPS-BAL. Genes with >10-fold difference in mean expression are shown. Genes discussed in the text are colored burgundy Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31040289), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Axl by Flow Cytometry

Inflammatory stimuli associated with viral infections upregulate Axl and promote Gas6 binding to macrophages. (A–E) MDMs were stimulated for 24 h with dexamethasone (Dex), LPS, IFN‐ gamma, poly(I:C) or IFN‐ alpha. (A) MerTK and (C) Axl protein expression determined by ELISA of total cell lysates (mean+SEM, n = 4). (B) MerTK and (D) Axl protein expression analyzed by western blotting; actin was used as loading control. A representative of two to three independent experiments is shown. (E) Flow cytometric analysis of Axl expression on MDMs stimulated with poly(I:C) or IFN‐ alpha for 24 h. Representative histograms of two independent experiments are shown; dotted line/shaded: FMO control. (F) Axl and MerTK relative mRNA and (G) protein expression in MDMs differentiated by 6‐day culture in M‐CSF or GM‐CSF determined by qPCR (mean+SEM, n = 6) and by ELISA of total cell lysates (mean+SEM, n = 3), respectively. (H) Axl, MerTK and Tyro3 relative mRNA expression in MDMs stimulated as in (A–C) analyzed by qPCR (mean+SEM, n = 2–3). (I and J) Flow cytometric analysis of Gas6 binding to MDMs stimulated with poly(I:C) for 24 h. (I) Representative of four independent experiments is shown; dotted line/shaded: isotype control. (J) Ratio of Gas6 to isotype control geometric mean fluorescence intensity (MFI)+SEM in unstimulated and poly(I:C)‐stimulated MDMs (n = 4). *p < 0.01, paired t‐test. ELISA and qPCR samples were assayed in duplicate. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29400409), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Axl by Flow Cytometry

Neutrophils and mononuclear phagocytes are expanded in the alveolar airspace following LPS inhalation. a Schematic overview of study design. Solid arrows denote LPS inhalation (red) or saline inhalation control (blue). Black arrows denote blood sampling. Dashed arrows denote BAL. b Flow cytometry of leukocyte preparations from SS-BAL, LPS-BAL, and HC blood. The CD45 versus SSC plot was used to define CD45+SSChi AM, CD45loSSCmid neutrophils and CD45+SSClo mononuclear cells (see also Supplementary Fig. 1). Monocyte/macrophages and DCs were negative for lineage markers CD3, CD19, CD20, and CD56 and expressed HLA-DR. Monocyte/macrophages were divided into CD14++CD16−, CD14++CD16+, and CD14-CD16++ populations analogous to blood classical, intermediate and non-classical monocytes. DCs within the CD14-CD16− gate were divided into subsets: Axl+Siglec6+ DC5s, CD11cloCD123+ pDCs, CD1c+BTLAlo-mid DC2/3s, and BTLAhi DC1s. Plots are representative of n = 9 SS BAL and n = 10 LPS BAL. c Concentrations of neutrophil, monocyte/macrophage and DC subsets in SS-BAL and LPS BAL. Bars represent mean and lines SEM. p-values from unpaired t-tests of SS versus LPS are shown: “ns” p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d Monocyte/macrophage and DC frequency in HC blood, SS-BAL and LPS-BAL as a proportion of SSClo MHC class II-expressing cells (not-including CD1c- DCs). e Concentration of selected leukocyte populations in peripheral blood at 2-h intervals following inhalation of saline (blue line) or LPS (red line). Data points show mean ± SEM for 3–5 participants. p-values from one-way ANOVA are shown. p-value representation is described in c Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31040289), licensed under a CC-BY license. Not internally tested by R&D Systems.